Puquitinib mesylate (XC-302) induces autophagy via inhibiting the PI3K/AKT/mTOR signaling pathway in nasopharyngeal cancer cells

Abstract

There are numerous studies that demonstrate the anti-neoplastic activity of phosphatidylinositol 3-kinase (PI3K) inhibitors and the mechanisms of inducing autophagy in cancer cells. The new anticancer drug puquitinib mesylate (XC-302) is a molecular-targeted drug, which suppresses the activity of PI3K directly. However, it remains unclear whether XC‑302 can develop an antitumor effect by inducing autophagy in nasopharyngeal cancer cells. The MTT assay was used to study the anti-proliferative effects of XC-302. Subsequently, autophagy was determined by monodansylcadaverine (MDC) staining, punctate localization of green fluorescent protein (GFP)-light chain 3 (LC3), LC3 protein blotting and electron microscopy. The expression levels of beclin 1, p62, protein kinase B (AKT), phospho (p)‑AKT, mechanistic target of rapamycin (mTOR) and p‑mTOR in XC-302‑induced autophagy were detected. Autophagy inhibition was assayed by 3-methyladenine (3‑MA) or small interfering RNA (siRNA) silencing of beclin 1. XC-302 inhibited the viability of CNE‑2 in a dose-dependent manner and the IC50 of 72 h was 5.2 µmol/l. After cells were exposed to XC-302 for 24 h, MDC-labeled autophagolysosomes were evident in CNE-2 cells by fluorescence microscope. Autophagosomes and autolysosomes were identified by transmission electron microscopy. Following transfection with GFP‑LC3, XC-302 induced a significant accumulation of GFP‑LC3, as monitored by a confocal microscope, which was reduced by 3-MA. XC-302 induced the formation of LC3‑II, increased beclin 1 levels and decreased the expression of p62. Additionally, the expression levels of p‑AKT and p‑mTOR were reduced with the elevation of XC-302. Knockdown of beclin 1 with siRNA or co-treatment with 3-MA enhanced significantly the survival of CNE-2 and promoted the ability of clone formation. XC-302 also induced apoptosis in CNE-2, and when autophagy was inhibited by 3-MA, the apoptosis rate was decreased. The present data provides the evidence that XC-302 can induce autophagy in CNE-2, which promotes the program of cell death and inhibits the PI3K/AKT/mTOR signaling pathway. Furthermore, XC-302 also promoted apoptosis in CNE-2 cells, which could be reduced when autophagy was suppressed, meaning that autophagy may interact with apoptosis to induce cell death.

Figure 1

The structural formula of XC-302. C₁₇H₁₅N₇•2CH₃SO₃H•2H₂O; Molecular weight: 545.59.

Figure 2

XC-302 inhibits proliferation, as well as induces apoptosis and autophagy, in CNE-2 cell lines. (A) After 24 h treatment of XC-302 (16–0.125 µM), CNE-2 cell viability was detected by the MTT assay. (B) XC-302 treated cells were stained with monodansylcadaverine (MDC), and the clearly visible images of staining were examined by a fluorescence microscopy. (C) CNE-2 cells were treated as described for 24 h and apoptosis was analyzed by flow cytometry following Annexin V-propidium iodide staning. (D) The formation of autophagosomes in XC-302-treated cells observed under transmission electron microscopy. The whole organelles could be observed in the cells in the untreated group. The XC-302 treatment (4 µM) not only resulted in the accumulation of autophagosomes (thick arrow), but also the formation of autolysosomes (thin arrow) in a portion of CNE-2 cells (magnification, ×37,000).

Figure 3

Western blot analysis was performed for autophagy- and phospha-tidylinositol 3-kinase (PI3K) pathway-related markers. (A) The cells were treated with XC-302 (8.0–0.5 µM) for 24 h. Cell lysates were prepared, and an immunoblot was performed using antibodies against LC3-I, LC3-II, beclin 1 and p62. (B) The effects of 4 µM XC-302 incubation on the protein expression of LC3-I, LC3-II and p62 in CNE-2 cells at 0, 3, 6, 12, 24 and 36 h. (C) Cells were treated with different concentrations of XC-302 for 24 h. The immunoblot was conducted and probed with anti-AKT, anti-p-AKT, anti-mTOR and anti-p-mTOR. LC, light chain; AKT, protein kinase B; mTOR, mechanistic target of rapamycin.

Figure 4

Apoptosis and autophagy in CNE-2 cells, induced by XC-302, which could be inhibited by exposure to 3-methyladenine (3-MA). (A) Testing of apoptosis was by Annexin V-FITC/propidium iodide assays, and the CNE-2 cells were treated by 4 and 8 µM XC-302 in the absence (left) or presence (right) of 5 mM 3-MA for 24 h. (B) The apoptosis rates are described as the mean of three independent experiments ± standard deviation. P-values were obtained by comparing the apoptosis rates in cells treated with XC-302 alone with those in 3-MA combined with XC-302 (^*P<0.05). (C) Images acquired by fluorescence microscopy, green fluorescent protein (GFP)-light chain 3 (LC3)-transfected cells treated with dose increasing XC-302 showed increased GFP-LC3 dots (upper), whereas at an XC-302 concentration of 8 µM, 3-MA significantly decreased the green fluorescence (lower panels).

Figure 5

3-Methyladenine (3-MA) accelerates the proliferation and clone formation of the CNE-2 cell line by suppressing autophagy. (A) CNE-2 cells were exposed to 4 µM XC-302/5 µM 3-MA, or a co-treatment of XC-302 and 3-MA for 24 and 48 h. The survival rate was compared by the MTT assay. (B and C) Cells were treated with 4 µM XC-302/5 µM 3-MA, or a combination of XC-302 and 3-MA for 6 h, and the clone formation ability was analyzed. Mean of three independent experiments; bars, mean ± standard deviation (^*P<0.05).

Figure 6

Stimulations of silencing beclin 1 on proliferation and clone formation in the CNE-2 cell line treated by XC-302. (A) CNE-2 cells were transfected with beclin 1 small interfering RNA (siRNA) or a non-target control siRNA for 48 h, and the expression of beclin 1 was verified by western blotting analysis. (B) The MTT assay measured cell viability between four groups (si-control, si-control cells with 4 µM XC-302, si-beclin 1 and si-beclin 1 cells with 4 µM XC-302). (C) Cells were treated with different drugs and siRNA, and the clone formation ability was analyzed. (D) The clone formation abilities are presented as the mean ± standard deviation. The P-value was measured by comparing the clone numbers between XC-302-treated cells silenced by si-control and those by si-beclin (^*P<0.05).